Histone deacetylase inhibition reduces deleterious cytokine release induced by ingenol stimulation

Abstract

Latency reversing agents (LRAs), such as protein kinase C (PKC) agonists, constitute a promising strategy for exposing and eliminating the HIV-1 latent reservoir. PKC agonists activate NF-κB and induce deleterious pro-inflammatory cytokine production. Adjuvant pharmacological agents, such as ruxolitinib, a JAK inhibitor, have previously been combined with LRAs to reduce deleterious pro-inflammatory cytokine secretion without inhibiting HIV-1 reactivation in vitro. Histone deacetylase inhibitors (HDACi) are known to dampen pro-inflammatory cytokine secretion in the context of other diseases and synergize with LRAs to reactivate latent HIV-1. This study investigates whether a panel of epigenetic modifiers, including HDACi, could dampen PKC-induced pro-inflammatory cytokine secretion during latency reversal. We screened an epigenetic modifier library for compounds that reduced intracellular IL-6 production induced by the PKC agonist Ingenol-3,20-dibenzoate. We further tested the most promising epigenetic inhibitor class, HDACi, for their ability to reduce pro-inflammatory cytokines and reactivate latent HIV-1 ex vivo. We identified nine epigenetic modulators that reduced PKC-induced intracellular IL-6. In cells from aviremic individuals living with HIV-1, the HDAC1-3 inhibitor, suberohydroxamic acid (SBHA), reduced secretion of pro-inflammatory cytokines TNF-α, IL-5, IL-2r, and IL-17 but did not significantly reactivate latent HIV-1 when combined with Ingenol-3,20-dibenzoate. Combining SBHA and Ingenol-3,20-dibenzoate reduces deleterious cytokine production during latency reversal but does not induce significant viral reactivation in aviremic donor PBMCs. The ability of SBHA to reduce PKC-induced pro-inflammatory cytokines when combined with Ingenol-3,20-dibenzoate suggests SBHA can be used to reduced PKC induced pro-inflammatory cytokines but not to achieve latency reversal in the context of HIV-1.

Keywords: Cytokine; HDACi; HIV-1; Ingenol-3,20-dibenzoate; Latency reversing agent; Suberohydroxamic acid.

Figure 1.. Epigenetic modifiers screening identifying compounds that dampen cytokine production induced by Ingenol-3,20-dibenzoate.

Screening of 96 epigenetic modifiers for compounds that reduced intracellular IL-6 induced by IDB in healthy donor PBMCs (n=1) revealed nine epigenetic modifier ‘hits’ that reduce intracellular IL-6 by fourfold or greater (at or below dotted line) when compared to IDB treatment alone. Ruxolitinib was included as a positive control for reduction of intracellular IL-6 when combined with Ingenol-3,20-dibenzoate. Compounds are listed by CAS number and color coded according to categorized function. All nine ‘hits’ were selected for further testing.

Figure 2.. A) SBHA reduces pro-inflammatory cytokine secretion induced by IDB ex vivo.

Solid lines represent the mean change in pro-inflammatory cytokine concentrations in the supernatant of PBMCs isolated from HIV-1 positive aviremic individuals (n = 8) and treated with SBHA (75nM or 100nM) and IDB (100nM) for 72 hours compared to IDB alone. The dotted line represents the baseline for the assay. *P value <0.05; **P value <0.01. B) Panobinostat has no significant effect on IDB induced pro-inflammatory cytokine production ex vivo. PBMCs from aviremic individual was treated with Panobinostat (100nM) and IDB (100nM). The mean change in pro-inflammatory cytokines is indicated by a solid line and dotted lines indicate the baseline for the assay.

Figure 3.. HIV-1 latency reversal induced by epigenetic modifiers alone or in combination with Ingenol-3,20-dibenzoate ex vivo.

Treatment of rCD4 cells isolated from HIV-1 positive aviremic individuals with Ingenol-3,20-dibenzoate significantly increased viral latency reactivation compared to media alone. The positive control αCD3/αCD28 resulted in viral reactivation in all donors. All nine epigenetic modifiers alone or in combination with Ingenol3,20-dibenzoate did not increase reactivation significantly compared to Ingenol-3,20-dibenzoate treatment alone. *P value <0.05; ** P value <0.01. The dotted line indicates the limit of detection of the assay (50 copies/ug cell associated RNA (caRNA)).